222 OP

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: other: clastogenicity/aneugenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: minimum 7 weeks
- Assigned to test groups randomly: yes
- Housing: 5 animals of identical sex per cage, IVC cage (Polysulphone), Type II L
- Diet (ad libitum): Altromin 1324 maintenance diet for rats and mice
- Water (ad libitum): tap water, sulphur acidified to pH value of approx. 2.8 (drinking water, municipal residue control, micro-biologically controlled at frequent intervals)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x per hour
- Photoperiod (hrs dark / hrs light): artificial light 6:00 - 18:00

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle/solvent used: 0.9% NaCl
- Justification for choice of solvent/vehicle: The solvent was chosen based on best solubility of the test item and according to its relative non-toxicity for the animals.
- Concentration of test material in vehicle: 2000 mg/mL (1 MTD), 1000 mg/mL (0.5 MTD), 400 mg/mL (0.2 MTD)
- Amount of vehicle (if gavage or dermal): 10 mL per kg body weight
- Lot/batch no.: 121678081 (Braun), 19FG3ÜWA (Fresenius Kabi)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The test item was suspended in 0.9% NaCl within 1 h before treatment. All animals received a single volume ip of 10 mL/kg bw.

Frequency of treatment:

The animals received the test item once ip.

Post exposure period:

Sampling of the bone marrow cells was carried out on animals 24 and 48 h after treatment.

Doses / concentrations

Remarks:

Doses / Concentrations:
three dose groups: 1 MTD (200 mg/kg bw), 0.5 MTD (100 mg/kg bw), 0.2 MTD (40 mg/kg bw)
Basis:
other: suspension in 0.9% NaCl

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control: clastogenic control substance, good stability at room temperature, broad basis of historical laboratory data
- Route of administration: intraperitoneal
- Doses / concentrations: 40 mg/kg body weight

Examinations

Tissues and cell types examined:

immature erythrocytes of the bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preceding study on toxicity was performed with the same strain and under identical conditions as in the main study. Three animals of each sex were treated ip for detection of the maximum tolerable dose. The maximum dose that was applied was 2000 mg/kg bw according the OECD 474 guideline. The maximum volume which was administered was 10 mL/kg bw. The highest dose group evaluated in the main experiment (2000 mg/kg bw) was based on the toxicity observed in the pre-experiment.

METHOD OF ANALYSIS:
All slides, including those of positive and negative controls were independently coded before microscopic analysis. Evaluation of the slides was performed microscopically by using 100 x oil immersion objectives. 2000 immature erythrocytes were scored per animal for the incidence of micronucleated immature erythrocytes. To detect an eventually occurring cytotoxic effect of the test item the ratio between immature and mature erythrocytes was determined. At least 200 erythrocytes were counted per animal and the result was expressed as relative PCE (rel. PCE = proportion of immature (polychromatic) erythrocytes among total erythrocytes).

Evaluation criteria:

There are several criteria for determining a positive result:
- dose-related increase in the number of micronucleated cells and/or
- biologically relevant increase in the number of micronucleated cells for at least one of the dose groups.
According to the OECD guideline, the biological relevance as well as the statistical significance of the results are the criterion for the interpretation.
A test item is considered to be negative if there is no biologically relevant and/or statistically significant increase in the number of micronucleated cells at any dose level.

Statistics:

For the statistics the nonparametric Mann-Whitney Test was used. However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals:
In the pre-experiment a concentration of 200 mg/mL (active component) of the test item was evaluated. Three male and three female mice received a single dose of 2000 mg/kg bw (active component) i.p. and showed toxicity such as reduction of spontaneous activity, constricted abdomen, piloerection, half eyelid closure, bradykinesia, recumbency, opisthotonos and catalepsis.

RESULTS OF DEFINITIVE STUDY
2000 mg/kg bw (active component) was tested as the maximum tolerable dose (1 MTD, 24 h and 48 h) in the main experiment. The volume administered i.p. was 10 mL/kg bw.
All animals treated with the highest dose (1 MTD, 24 h and 48 h) showed moderate toxic effects. Animals treated with 1000 mg/kg bw (0.5 MTD) showed the same signs of toxicity as displayed for the 1 MTD dose group animals, except opisthotonos, recumbency and ataxia. The signs of toxicity were in total less intensely developed (mild/moderate) than in the 1 MTD group animals. After 4 h no toxic symptoms were observed anymore. The animals treated with 400 mg/kg bw (0.2 MTD) showed the same toxic effects as mentioned for the 0.5 MTD dose group. The signs of toxicity were mildly expressed. After 3 h no toxic symptoms were observed anymore for the 0.2 MTD female dose group and after 4 h no toxic symptoms were observed anymore for the 0.2 MTD male dose group.


- Induction of micronuclei (for Micronucleus assay):
The negative controls (24 h and 48 h) evaluated were within the range of the historical negative control data (0.04 – 0.29%). The mean values of micronuclei observed for the negative control (24 h) were 0.27% (male mice) and 0.17% (female mice). The mean value for the 48 h negative control was 0.10% (male and female mice).
The dose group treated with 0.2 MTD showed mean values of 0.15% (male mice) and 0.27% (female mice). The value observed in the female group was increased compared to the corresponding negative control, but this increase was not statistically significant. The value observed in the male group was decreased compared to the corresponding negative control, but this decrease was not statistically significant. Additionally, both values were within the range of the historical negative control data.
The dose group treated with 0.5 MTD showed mean values of 0.15% (male mice) and 0.11% (female mice). The values observed in the male and female group were reduced compared to the corresponding negative control, but these decreases were not statistically significant. Additionally, both values were within the range of the historical negative control data.
The dose group treated with 1 MTD (24 h treatment) showed mean values of 0.17% (male mice) and 0.13% (female mice). The values observed in the male and female group were reduced compared to the corresponding negative control, but these decreases were not statistically significant. Additionally, both values were within the range of the historical negative control data.
The mean values observed for the 1 MTD 48 h treatment were 0.15% (male mice) and 0.06% (female mice). The value observed in the male group was increased compared to the corresponding negative control and the value observed in the female group was decreased compared to the corresponding negative control, but this increase/decrease was not statistically significant. Additionally, both values were within the range of the historical negative control data.
No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.



- Ratio of PCE/NCE (for Micronucleus assay):
The negative controls (24 h, 48 h) were within the range of the historical negative control data (0.46 - 0.79). The mean value noted for the 24 h negative control was 0.68 (male and female mice). The mean values detected for the 48 h negative control were 0.58 (male mice) and 0.60 (female mice).
The animal group treated with 0.2 MTD showed mean values of the relative PCE of 0.62 (male mice) and 0.50 (female mice). The mean values observed in the male and in the female group were slightly decreased as compared to the corresponding negative control. However these decreases were not statistically significant. Moreover the mean values were within the range of the historical control data.
The dose groups which were treated with 0.5 MTD showed mean values of the relative PCE of 0.57 (male mice) and 0.51 (female mice). The mean values observed in the male and in the female group were slightly decreased as compared to the corresponding negative control. However these decreases were not statistically significant. Moreover the mean values were within the range of the historical control data.
The animals who received 1 MTD (24 h treatment) showed mean values of 0.64 (male mice) and 0.44 (female mice). The mean values observed in the male and in the female group were decreased as compared to the corresponding negative control. However these decreases were not statistically significant. Moreover the mean values were within the range of the historical control data.
The animal group which was treated with 1 MTD (48 h treatment) showed mean values of the relative PCE of 0.54 (male mice) and 0.56 (female mice). The mean values observed in the male and in the female group were slightly decreased as compared to the corresponding negative control. However these decreases were not statistically significant. Moreover the mean values were within the range of the historical control data.


- Statistical evaluation:
The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant differences (p<0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
In conclusion, it can be stated that during the study and under the experimental conditions reported, the test item did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.

Executive summary:

In a mammalian micronucleus test of murine bone marrow cells, five male and female animals per dose group were treated ip with the substance at doses of 2000, 1000 and 400 mg/kg bw.  Bone marrow was obtained from the femurs (after animals were sacrificed by cervical dislocation) at 24 h (all dose and control groups) and 48 h (negative control and 1 MTD group) post-treatment.  The vehicle was 0.9% NaCl. The animals received the test item once ip.
There were signs of toxicity during the study.  The animals treated with doses of 0.2 MTD, 0.5 MTD and 1 MTD showed dose-depended mild to moderate signs of systemic toxicity. The animals treated with 1 MTD showed moderate signs of systemic toxicity such as reduction of spontaneous activity, constricted abdomen, bradykinesia, opisthotonos, piloerection, ataxia, recumbency and half eyelid closure. The substance was tested at an adequate dose based on OECD 474. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood cells after any treatment time.
This study is classified as acceptable and was performed in order to be compliant with Chinese regulations for notification. This study satisfies the requirement for Test Guideline OPPTS 870.5395; OECD 474 for in vivo cytogenetic mutagenicity data.